Stroke Researchers

Dr. Vardan Karamyan

  Prior to meeting with Professor Karamyan, the EmpireGene team was researching neurolysin, mitochondrial, a protein that is encoded by the NLN gene. Previous research attempting to elucidate the mechanisms of NLN found that in the acute phase after stroke, neurolysin was upregulated in mouse models of the brain. NLN is linked to the human brain’s self-preserving and self-repair mechanisms after stroke. NLN is a multi-pathway target, which makes it more desirable for the pharmacotherapy of stroke than just one factor. This is important in the context that the brain has well-developed, complex, and highly conserved endogenous mechanisms for response to stroke and traumatic brain injury. Professor Vardan Karamyan is an associate professor for the School of Pharmacy at the Texas Tech University Health Sciences Center. His research career has focused on the role of neurolysin and peptidases in the brain. We met with Professor Karamyan on March 28th for 30 minutes. He explained his research on neurolysin and neural repair and helped to guide our research focus. Notably, Professor Karamyan recommended that we avoid researching neurolysin. Professor Karamyan noted that neurolysin does not have a significant effect on the brain’s neural repair. However, Professor Karamyan did recommend other pathways for research. For instance, he encouraged us to research other biomarkers in stroke recovery that can distinguish which patients will or won’t improve from some kind of therapy. He also suggested researching a protein that could boost the neuroplasticity of and improve connections between neurons in the brain. Professor Karamyan also identified mRNA technology, which has been critical for COVID-19 vaccines, as a potential method for protecting neurons after stroke; there has already been some research on this. Notably, Professor Karamyan strongly recommended that for our iGEM research project, we bioengineer a protein to cross the Blood-Brain Barrier. 98% of all small molecules are unable to pass through the Blood-Brain Barrier, which has stalled the use of stroke-recovery drugs. However, a molecule like a protein can pass through the Blood-Brain Barrier through a Trojan horse method.

Dr. Michelle Cheng

  We interviewed Dr. Michelle Cheng, a senior research scientist who acquired a PhD in Neuropharmacology from University of California. We were still within the early phase of deciding our research topic, and we wanted to learn more about optogenetics, next-generation sequencing, molecular & cellular biology, immunofluorescence, in-vitro and in-vivo injury models. Interviewing her helped us in finding our research topic as her major interest was about studying brain repair and recovery at both the neural circuit and molecular level in order to develop strategies that promote the recovery process.

Here are some questions asked and answered.

Ashley Son: “How would optogenetic neuronal stimulation promote post stroke recovery?”
Professor Cheng: “The reason scientists use optogenetic neuronal stimulation is because it can effectively isolate the factors that contribute to remodeling and recovery processes after the stroke. Using Optogenetics, they can selectively manipulate the excitability of specific cell groups with millisecond-scale temporal precision in a manner more similar to endogenous neuronal firing patterns. The mechanism is not fully identified in the course of current biogenetics, but light-activated microbial proteins like Channelrhodopsin 2 (ChR2) –which depolarizes neurons when illuminated with blue light–or Halorhodopsin (NpHR) –which hyperpolarizes neurons–contribute to the identification process”.
Ashley Son: “What are effective ways to stimulate neural stem cell proliferation?”
Professor Cheng: “There are different ways for a patient's stem cells to proliferate. Some examples are applying intermittent fasting, caloric restriction, reducing triglycerides, and taking supplements like vitamin D, resveratrol, curcumin or turmeric supplements.”
Amanda Son: “What are the obstacles in post-stroke neural repair research?”
Professor Cheng: “The major difficulty is identifying whether the mice are truly suffering from stroke or not. Also, a lack of prior stroke repair research is a big problem. As for now, tissue plasminogen activator is the only drug clinically approved to help stroke patients despite thousands of potential therapies.”
Rae Kim: “What are the drawbacks of the Trojan horse method when crossing the Blood-Brain Barrier?”
Professor Cheng: “Trojan horse crossing significantly contributes to fungal barrier crossing and that host factors regulate this process independently of free fungal transit. It also enables CNS entry of fungal mutants that cannot otherwise traverse the BBB. The route of Trojan horse injection was never scientifically identified and therefore, there is no assurance about the neuronal damage it costs.”

Dr. Vardan Karamyan

  As we finished engineering our final recombinant plasmids, our team organized another meeting with Dr. Karamyan. At this point in the competition, we were trying to complete the remainder of our research within a 3-week time window. Dr. Karamyan encouraged us to consider how much time it would take for us to run the trans-well and interaction assays. He advised that we skip the purification step after engineering our HIRMAb-NT3 and HIRMAb-FGF2 proteins, which we followed. Overall, Dr. Karamyan was impressed by our research progress, and agreed that our research was relevant within the field of Trojan horse repair. He did, however, bring up that he wanted us to consider potential side effects from artificially upregulating NT-3 and FGF-2 in the brain. He brought up that he read a research paper that found NGF (Nerve Growth Factor) levels to be increased in preclinical models of inflammation and peripheral nerve injury. Clinically, NGF concentration is also increased in chronic pain conditions such as interstitial cystitis, prostatitis, arthritis, pancreatitis, chronic headaches, cancer pain, diabetic neuropathy, and noncancer pain. NT-3 is in the same protein family, which implicates NT-3 as a potential cause for chronic pain. This would mean that while our therapy is meant to rehabilitate neurons, it might activate certain pathways that have negative consequences on the brain. After meeting with Dr. Karamyan, our team decided to expand our human practices outreach to include regulators within the FDA as well as public health officials, who may have more answers to some of our questions on our therapy's potential side effects.

Dr. William Costain

  In September, we had a meeting with Dr. Costain, who is a research scientist at the National Research Council in Ottawa, specifically in the area of brain delivery of drugs and repairing brain diseases. We inquired about potential safety concerns with HIRMAb and side effects of the delivery of these therapeutic proteins to neurons. Dr. Costain replied that he was not too concerned safety-wise. Furthermore, Dr. Costain also stated that a therapy more complex than NT-3 or FGF-2 could be delivered into the brain, including Receptor Mediated Transcytosis (RMT) mediated delivery of nanoparticles and enzyme replacement therapy. The complexity of what we deliver is not limited by the RMT mechanism, which makes it possible for a variety of varying sizes of material to cross the BBB. We also asked about other therapeutic options for drug delivery across the BBB, such as focused ultrasound. Dr. Costain replied that focused ultrasound can only affect a particular part of the brain, which may be helpful in such disease. However, when targeting the entire brain, the RMT mechanism would be a more appropriate option. Meeting with Dr. Costain affected how we viewed our project in the long term, as he identified that HIRMAb could interact with other drugs when crossing the BBB.

Clinical Doctors

Dr. Imama Naqvi

  During the summer, our team organized an in-person meeting with Dr. Naqvi at the Columbia University Medical Center's Division of Stroke and Cerebrovascular Disease. We discussed our plans for our wet-lab experiments, and Dr. Naqvi advised that we use SH-SY5Y cells as a model of stroke, starving them of nutrients for a temporary period of time and then reintroducing the cell culture media. We could then compare inflammatory markers and free radicals when the fusion protein is reintroduced within media and when it isn't. Dr. Naqvi clarified some of our questions about existing stroke therapies, and highlighted that currently, there is a limited set of therapy options within the acute phase (less than 3 months) of a stroke, including tinectaplase and endovascular treatment. Dr. Naqvi also mentioned that outside of stem cell therapy, there is nothing that aims to improve neuron function afer a stroke. Thus, our therapy could be the first alternative to this form of therapy. Overall, Dr. Naqvi was supportive of our research idea, and encouraged us to propose NeuroTrojan as a therapy to be used in conjunction with the existing ones that remove stroke-causing clots. Our most important takeaway from the meeting was that NeuroTrojan would be most effectively delivered through transvascular injection, which improved our proposed implementation.

Dr. Jeffrey Greenfield

  Our team met with Dr. Greenfield due to his prior experience in brain tumors and intra-arterial delivery systems. Due to our prior conversation with Dr. Naqvi, we concluded that an intra-arterial system may be the most effective in carrying our Trojan Horses through the Blood-Brain Barrier (BBB). Our discussion, however, did not conclude that intra-arterial was the best method. We discussed the advantages of simply using our Trojan Horses to cross the barrier to ensure the fastest and safest delivery. Other mechanisms, such as mannitol, that fully open the barrier could lead to precarious infections. Dr. Greenfield’s research highly utilized microcatheters, which have been proven to be very safe and were studied in clinical trials before integrating them in applicable solutions to real human patients. One novel option we did discuss was the integration of a balloon occlusion. This would allow our delivery system to be more targeted, but again we are continuing with a method as least invasive as possible. Dr. Greenfield also assuaged us of many of our concerns, particularly Brownian motion. We were concerned that once our Trojan Horses had passed through the BBB, they would not all attach to the neurons. However, based on Dr. Greenfield’s previous research and exposure, he does not believe this would be problematic as many other drugs use the same facilitation method as our Trojan Horses and are still able to attach to their cell. Lastly, we discussed future implementation of our work - the next steps needed in order to make our cure a viable option for healthcare. As of right now, we are working in a laboratory and are using a hCMEC/D3 cell line in order to simulate the actual process of the BBB. In order to test this on real subjects, we could first begin with animal models. In order to regulate the distribution of our proteins, we could also attach modifiable agents in order to monitor. We can also utilize real time imaging with PET scans to see the diffusion of our proteins. If our methods are proven to be successful, we can even consider making this Trojan Horse method popular for other types of brain injuries such as ALS.

Dr. Norman Pflaster

  Dr. Pflaster treats stroke victims everday by prescribing thrombolytic therapy or referring them to other people to perform thrombectomy. He claims that Thrombolysis is more effective than aspirin or plavix especially in patients who are “appopiate candidates.” According to Dr. Pflaster, the first symptoms he sees in stroke patients are focal nuerologic symptons which can sometimes be mild. The biggest problems patients face are complete weakness, aphasia, coma, death, or a persistent vegetative state. For patients who have not recovered from a stroke, medical support is needed to keep them alive aswell as nutritional support and then rehabilitation for motor, functional and cognitive issues. Some things that can prevent patients from making a straightforward recovery are the severity of the stroke, hemorrhage into the stroke, infection during recovery, psychosocial complications, malnutrition, falls, or other medical complications. With regard to the implementation of our delivery method for a therapeutic Trojan horse which would likely be a pill or a transvascular inejction, Dr. Pflaster states that the optimal delivery mechanism would be a vascular injection or intrathecal delivery. However, the limitations would be the penetration of the blood-brain barrier and failure of circulation in the area of ischemia. Dr. Pflaster further sates that if this method has neuroprotective benefits, it would be beneficial to people who had smaller strokes.

Patient & Caretaker Interviews

  Our team had the valuable experience of meeting with a victim of hemorrhagic stroke. On September 20th, we had a zoom meeting with them (identity kept private), and they answered some questions about their experience and recovery with the stroke. Initially, they were given no treatment, and it took multiple hospital visits along with chronic pain reports to receive real medical attention. They eventually received a VP shunt placement and an aneurysm clipping; this resulted in impeded speech from right frontal lobe damage. Their personal life suffered greatly due to their state of speech and inability to go back to work. It took lots of cognitive and emotional therapy to get where they are today and the process was long and draining. Currently, they take ADHD medications (which they took before the stroke too) however they stopped during more difficult periods of their treatment. Now, they use them to treat executive functions struggles like time management or attention span. The medications can result in migraines or headaches - mainly a result of stress and sleep - but that is also a long-term result of the stroke itself. The victim talked to us about her experience finding a job after her stroke, she mentioned severe difficulty with maintaining the job especially as the regulations in her state make receiving a disability label hard to obtain. They are now a local activist that fights against bills and practices that impede the daily life of disabled individuals. Their insight was helpful in understanding how damaging strokes can be to normal people’s life and why it is so important to conduct research on post-stroke recovery.

  At the Little Neck Care Center, we asked one of the aides if we could interview one of the patients. We were lucky enough to be able to interview a patient in person. When we asked him if he remembered the first time that he was diagnosed with a stroke, he told us that he had already had four strokes at the point where he first took the MRI scan. When we asked if he had any symptoms until he was diagnosed, he said he did not have any symptoms. However, when he discovered that he had strokes, he started to lose balance. He has been taking physiological therapy for two years, but it has not made any significant changes, and he was annoyed by the fact that a cure did not exist for such a common ailment. When we gave him details on what our project aimed to solve, he thought that we had a good concept, and he wished us luck. Our most interesting takeaway from the interview was that physiological therapy is a limited method of supporting patients, thus showing the value of our project in its potential to rehabilitate neurons.

FDA Regulation

Dr. Julia Barrett

  Our iGEM team had a very valuable meeting with Dr. Julia Barrett, the Executive Vice President of Drug and Biological Products at Greenleaf Health. Due to her prior experience with the FDA and her knowledge of clinical regulations, she was a huge asset to our team’s progress. After our discussion with Dr. Barrett, we concluded that our fusion protein would be broken down by the human immune system and we would likely need to administer the protein multiple times for the effects to actually last. If the fusion protein is to be tested (not in clinical trials), the fusion protein would have to be tested on larger animals (rather than smaller ones) due to the fact that the arteries of larger animals are closer to the size of a human. We would also need to measure the amount of the fusion protein that crossed the Blood-Brain barrier to determine how successful it was. Dr. Barrett’s work with clinical regulations and the FDA put her in the position to provide knowledge of the process of getting a drug or biologic approved by the FDA. After evidence that a protein is successful in animals, it is time to move on to the first phase of clinical trials. This phase would entail anywhere from 10 to 50 healthy volunteers or post-stroke patients. Among these volunteers, there would be variable dose quantities and constant safety monitoring. Phase two usually has anywhere from 50 to 300 participants, most of which are the target audience of our protein. A control group and groups with various dosages help determine the extent that the drug helps the individual. The goal of this phase is to expand safety data as well as the preliminary ability to replicate the desired results. Phase three is focused on increasing the sample size, which by doing so increases the safety of the drug that is used because the drug has been cleared by more people. Julia Barrett mentioned there are many other phases that are not necessarily part of the clinical trial but part of the approval process. For example, there is the Pre-IND phase, which comes before any trials but includes the product concept, and determines the animal for the initial trial, as well as research and product development. Once the trial is complete, researchers need to complete a write-up that is then examined by the FDA in-depth over the course of 30 days. After the trial they submit a license application that takes 10 months to review and if it is approved, the researchers receive licensure.

Ms. Elizabeth Jungman

  We met with Elizabeth Jungman and Stefanie Kraus, knowledgeable FDA employees, to discuss the implementation of our solution. They have dealt with many companies with neurological drugs and were able to advise us through our research stages. Our research would involve three main stages - the cell culture, the animal models, and finally the human testing. The animal studies ensure that the drugs are not toxic to humans and must show successful results before taking the risk in human patients. The FDA typically becomes involved with a project during the human stage and conducts a pre-investigation of the drug. We also became informed of just how transparent the FDA is concerning product approval through the information available on the Department of Drug Information’s page. They also have a vast supply of information for small businesses, as their proposals are typically not as sophisticated. Many of these smaller businesses also utilize consultants to prepare their proposals to pass FDA inspection. The FDA looks through several features of the drug including toxicology and pharmacology before approval. A drug sponsor typically proves to the FDA that the drug is high quality and provides all applicable information. Batch consistency concerning the drug manufacturing is also important to prove, as all the drugs manufactured need to be guaranteed to have the same composition. All must be made by a specific process that is thoroughly researched into and this process must be repeated with every reproduction. The FDA does have some typical issues with project proposals, including deliberating whether the trials are well designed and whether they provide reliable information about the project. Often, there are issues with manufacturing data and statistical analysis. If there are adverse effects, the FDA employs a safety surveillance program and continues to monitor the project with a risk evaluation. In terms of risks versus benefits in a product, they implied that it requires a lot of moral debate. They monitor whether the side effects can be managed, how many patients have effects on the drug, and how severe any side effects are. They also recommended speaking to patients and determining what is most important to them and how they weigh the costs versus the benefits.

Care Center Performances

Little Neck Care Center

  At Little Neck Care Center, Ryan Oh introduced the EmpireGene team to the senior citizens of the Little Neck Care Center. He described the EmpireGene team's mission to find a treatment for the issue of post-stroke neural repair. Furthermore, he told them that the team would be giving them a musical performance, as music therapy is understood to help stroke patients through mood regulation, improved concentration, and promoting changes in the brain to improve function. After the opening speech, Ryan started with a piano performance, presenting Canon in C, the Main theme of "Spiderman: No Way Home", and Moonlight Sonata 1st movement. After Ryan, Joseph Jeon performed Sarabande in G Minor by Carl Bohm on the violin. At the end of the performance, Brigid Allen delivered a beautiful performance of two violin pieces: Bruch Violin Concerto No. 1 in G Minor, Movement 1 and Bach Partita Allemande.

Walter Strauss Stroke Center

  We gave a virtual performance at Walter Strauss Stroke Center. The event started with a strong opening by Jaimie Koh, who played several classical pieces on her clarinet, including Merry Go Round of Life by Joe Hisaishi. It was then followed by many of the original musicians who played at Little Neck Care Centers, along with poetry readings and singing performances by our talented team members.

Private Sector Interviews

Xylyx Bio

   As we refine our research, our team thought about the practicalities to implementing our fusion protein drug to the real world. As such, we sought the expertise of Mr. O’Neill, the co-founder of Xylyx Bio, a company dedicated to making drug discovery more accurate and actionable by bringing insight into the efficacy of candidate compounds. Mr. O’Neill gave us advice as to the process of how to get your drug from the lab to the market and how to best approach the FDA. He explained to us the phases of clinical trials. Phase 1 clinical trial assesses whether giving this drug in a typical dose (dose relevant for therapy) is safe and the threshold to its safety. For our fusion protein in particular, phase one consists of being asked to see all of our pre-clinical data such as animal testing, modeling, and lab testing on human cells. Once we have designed the study and have gotten approval, we move onto phase 2 otherwise known as the pivotal trials. Mr. O’Neill explained that in these phases, FDA focuses on ethicacy and tests for the appropriate dose and the thresholds for the therapeutic benefit. For instance, questions such as whether the side effects outweigh the maximum therapeutic benefit would be asked and tested. The third phase is implementation where larger cohorts of patience can be used and there may be different basis of comparisons. Through Mr. O’Neill’s valuable insight, our team is able to understand the process from lab to market much easier as we create and prepare our fusion protein. At the time of this interview, our team was also in the process of researching out to the Federal Drug Administration’s Center for Drug Evaluation and Research. Mr. O’Neilll advised us to reach out to GreenLeaf Health and M Squared Associates, two firms that specialized in consulting for companies approaching the FDA.

Dr. William Tanner

  We met with William Tanner, who is a titan of his industry. He helps various companies obtain FDA approval and was invaluable in discussing any flaws in our current project implementation. He first led us through the steps of testing - beginning with animals. Rodents are a prime subject for researching many drugs and if the trials are successful, provide great evidence for continuing the trial in human patients. He did inform us of some other biotechnological solutions, such as kinase-inhibitors for the regeneration of neurons, and led us through their approval process. This solution, while somewhat similar to ours, provides a very different approach. Tanner stated, however, that as long as our solution could provide a high-functioning option for buyers it would still be worth pursuing. The solutions for post-stroke neural repair are varied enough to provide clients with more options to choose from, and potentially a more successful method of assisting these neurons. In terms of the drug’s risk/benefit analysis, he views it as a question of whether the hazards are less serious than the disease being left untreated. In our case, we were concerned as one of our proteins was implicated in chronic pain. However, based on the nature of the problem we are intending to solve, this side effect may be considered less of a deterrent to the drug’s overall approval. One of the major concerns he mentioned is acquiring investors. Without investors, it is nearly impossible to develop a drug, given the infrastructure needed for its mass production. Investors mainly look for the return provided if they do provide their services. In order to enlist their support, it is essential to highlight the drug’s proven efficacy and to show the negative consequences for patients without a solution such as the one we are providing.